title: Dissecting key residues in folding and stability of the bacterial immunity protein 7.
abstract: The small four-helix immunity protein, Im7, has previously been shown to fold via a compact intermediate containing three of the four native helices. The short, six-residue helix III only docks onto the developing Im7 structure after the rate-limiting second transition state has been traversed. Previous work demonstrated that mutation of the helix III sequence can be used to trap the protein in the on-pathway intermediate ensemble at equilibrium. Here the role played by individual residues in the native helix III sequence in locking Im7 into a stable native structure is further examined. This work commenced with an Im7 sequence trapped in the partially folded state by substitution of the six residues in helix III with a polyglycine sequence. Biophysical analysis of variants in which individual residues from the native helix III sequence, and combinations of these residues, were introduced into this background demonstrated a critical requirement for three residues, Leu 53, Ile 54 and Tyr 55, to lock Im7 into its unique native structure. The results demonstrate a stringent constraint on the evolution of the Im7 helix III sequence rationalizing its high-sequence identity in the fold family. Thus, Leu 53 and Ile 54 provide crucial stabilizing interactions in the hydrophobic core of native Im7, while Tyr 55 is required for both stability and function. In contrast, Tyr 56 is critical for colicin binding and has no role in maintaining a stable native fold.
authors.affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Stuart
authors.initials: S
authors.lastname: Knowling
authors.forename: Alice I
authors.initials: AI
authors.lastname: Bartlett
authors.forename: Sheena E
authors.initials: SE
authors.lastname: Radford
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D058977:Molecular Sequence Annotation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D055550:Protein Stability; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: D002352:Carrier Proteins; C509253:E colicin-binding immunity protein Im7, E coli; D029968:Escherichia coli Proteins